Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K20/00—Accessory food factors for animal feeding-stuffs
  • A23K20/10—Organic substances
  • A23K20/179—Colouring agents, e.g. pigmenting or dyeing agents
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
  • A23L17/00—Food-from-the-sea products; Fish products; Fish meal; Fish-egg substitutes; Preparation or treatment thereof
  • A23L17/60—Edible seaweed
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S426/00—Food or edible material: processes, compositions, and products
  • Y10S426/807—Poultry or ruminant feed

Definitions

• the present invention relates generally to pigmentation supplements for animal feed compositions, and more particularly to the use of comminuted Haematococcus alga as a pigmentation supplement in
• compositions for feeding aquatic and other animals are 10 compositions for feeding aquatic and other animals.
• the artificial food sources should mimic the natural food sources so that the cultivated animal product closely resembles the naturally-harvested animal product.
• the present invention is concerned primarily .
• ⁇ ⁇ derive their natural pigmentation from a variety of carotenoids, such as beta-carotene, canthaxanthin, zeaxanthin, astaxanthin, astaxanthin ester, and the like.
• carotenoids such as beta-carotene, canthaxanthin, zeaxanthin, astaxanthin, astaxanthin ester, and the like.
• astaxanthin which provides a source of 5 pigmentation in a wide variety of aquatic animals, often without the need for the animal to biologically convert the astaxanthin to any other form of carotenoid.
• pigmentation supplement compositions comprise comminuted Haematococcus alga prepared by grinding dry encysted Haematococcus at cryogenic temperatures, to an average particle size below about 10 ⁇ i ⁇ .
• the comminuted particles will usually be treated to inhibit degradation by combination with an anti-oxidant or by various coating processes, such as gel coating, microencapsulation, oil coating, and the like.
• Such pigmentation supplement compositions have been found to be particularly suitable for incorporation in animal feeds, particularly aquatic animal feeds, in order to enhance pigmentation.
• Use of the encysted Haematococcus cells as a feed supplement requires that the cells be effectively fractured in order to provide a digestible product.
• Cryogenic grinding is superior to other comminution methods, such as enzyme treatment, which require separate wash and dry steps to remove enzyme from the product and which potentially leave an enzyme residue in the final product.
• the present invention allows preparation of final product in a single grinding step which does not require chemical or enzymatic treatment.
• the anti-degradation treatment allows for storage of the product, even over relatively prolonged periods . hen exposed to oxygen and light, without substantial degradation of the carotenoids.
• the comminuted Haematococcus cells can also serve as a source for extracted and purified carotenoids, particularly astaxanthin.
• Fig. 1 is a graph illustrating the comparative loss of xanthophyll between as samples containing an anti-oxidant and one not containing an anti-oxidant.
• the feed compositions of the present invention are prepared from encysted algal cells .of the genus Haematococcus .
• the genus Haematococcus consists of flagellated unicellular members of the green algae (Chlorophyceae) .
• the diagnostic characteristic distinguishing members of this genus from other members of the order Volvocales is the cell wall.
• the wall In flagellated cells, the wall is separated from the plasma membrane, being connected to it only by a series of cytoplasmic strands. Upon encystment, a new cell wall is formed inside the old cell wall. This cell wall thickens and becomes impervious to many types of chemical and physical stresses, making it refractory to conventional grinding techniques.
• the taxonomy of Haematococcus and its related genera is somewhat indistinct. Haematococcus was for a time referred to as Sphaerella , and differences with members of the genus Chlamydomonas are not always clear.
• Stephanos phaer a is a closely allied genus in which the cells grow in a colonial habit.
• Haematococcus species and strains which produce appreciable amounts of astaxanthins are suitable for use in the present invention.
• the amount of astaxanthin produced will be greater than 0.5% by weight of the alga on a dry basis, -more usually being at least about 1%, and desirably being 1.5% or greater.
• identified species and strains meeting these requirements include H. pluvialis , particularly H. pluvialis HI, H. pluvialis H2, H. pluvialis spitzbergenensis, and H. pluvialis tvaerminnensis; H. capensis, particularly H. capensis borealis; H. droebakensis, particularly H.
• Haematococcus is a common organism in nature and isolation of suitable new strains is well within the skill of the art.
• H. pluvialis which is characterized by both rapid growth and proficient production of astaxanthin.
• Particularly preferred is the use of H. pluvialis H2, available from Scripps Institute of Oceanography, La Jolla, California.
• the strain of Haematococcus selected for production will be maintained in an axenic stock culture with a reserve supply of encysted cells in case the stock culture is lost.
• a starter culture will be derived by expanding the stock culture in a defined media suitable for algal growth, such as half-strength Bold's basal media, preferably supplemented with thiamine, urea, and sodium acetate. The cells are grown in the stock culture to a density of about
• the culture media should be maintained at a pH in the range from about 6.5 to 8.
• the starter culture should be expanded until a sufficient volume of inoculum has been obtained for transfer to the production phase, typically in the range from about 50 to 500 L, usually in the range from about 100 to 200 L. Intermediate inoculum cultures may subsequently be grown.
• production ponds will have a volume in the range from about 50,000 to 1,000,000 L, more usually in the range from about 30,000 to 500,000 L.
• the production ponds may be located indoors or outdoors, with indoor locations being advantageous as they limit the potential for introduction of competing microorganisms. Outdoor locations are, of course, much less expensive to provide.
• 5 production in open ponds will usually be accomplished by batch procedures. After cleaning and decontaminating, the pond is filled with fresh water, typically of irrigation quality or better.
• the water will usually be treated with a sterilant, such as 0 chlorine, ozone, or ultraviolet light in order to retard the growth of competing organisms which may be present initially in the water. Suitable nutrients are then introduced'to the water.
• a nitrogen source such as ammonia or nitrate
• a 5 phosphorus source such as a phosphate
• the inoculum can be added.
• the volume of the inoculum provided will depend 0 on the volume in the production phase, usually being in the range from about 0.5 to 5% of the production volume, more usually being in the range from about 1 to 2% of the production volume.
• the production growth media should be
• Carbon dioxide gassing will typically be used to control pH as well as provide inorganic carbon for growth. Maximal growth is obtained when the cells are exposed to relatively low light conditions.
• heterotrophic cell growth may be achi.eved (i.n addi.tion to" autotrophic) by supplementing the growth medium with organic carbon sources, nitrogen sources, and vitamins.
• organic carbon sources are available, with acetic acid being preferred.
• Urea is the preferred nitrogen source and
• a desired cell density typically in the range from about 10 to 10 cells/ml, more typically in the range from about 3x10 5 to 6x105 cells/ml in autotrophic cultures.
• encystment of the algal cells will be promoted, typically by nutrient deprivation, an increase in salinity, or both.
• Nitrogen deprivation and/or salt (NaCl, CaCl 2 , and the like) concentrations above about 50 mM (0.3% by weight) have been found to promote encystment.
• the encysted cells may be harvested by cessation of pond mixing, allowing the cells to settle. Thereafter, the paste is heated to a temperature above about 70°C to dry the cells and kill the cells and any contaminating microorganisms. Optionally, the dried cells may be washed to remove extraneous material. Depending on the purity desired, further cleaning of the encysted cells may be appropriate, such as by washing with dilute acid.
• the dried, cleaned Haematococcus cells will be comminuted to form a powder having an average particle size below about 10 ⁇ m, preferably being below about 5 ⁇ . Particles in this size range are particularly suitable for incorporation into animal feed compositions, as will be described in more detail hereinafter.
• the powder After comminution, the powder will normally be treated to inhibit degradation of the carotenoids which are the desirable components.
• the particles of the powder may be coated with an edible material to form an oxygen barrier to inhibit oxidation.
• suitable gel coating, oil coating, and microencapsulation techniques are described in the patent and scientific literature.
• a sufficient amount of a suitable anti-oxidant may be added to inhibit degradation of the carotenoids present in the ground product.
• suitable anti-oxidants include butylated hydroxytoluene (BHT) , ethoxyquin, tocopherols, butylated hydroxyanisole, di-tert-butyl-paracresol, propyl gallate, and the like.
• BHT butylated hydroxytoluene
• the amount of anti-oxidant will depend on the particular anti-oxidant chosen, typically being in the range from about 0.05 to 5% by weight of the final product, more typically being in the range from about 0.1 to 3% by weight of the final product.
• the anti-oxidant may be added either before or after comminution of the alga. By adding before comminution, a separate mixing step may be avoided.
• the comminuted Haematococcus cells may be packaged in a manner which inhibits oxidation, such as vacuum packing or packing with oxygen absorbers.
• Such packing is not preferred, however, since the product will degrade as soon as the packaging is opened. .
• the method of grinding is critical to the present invention.
• the cells must be dry, in order to allow fracturing by high speed impact mills and jet mills. It has been found that grinding under cryogenic conditions, typically at a temperature below about -50°C, more typically at a temperature below about -170 ' C, is greatly facilitated and provides a highly uniform and well-preserved product.
• the encysted Haematococcus cells may be combined with a cryogenic liquid, such as liquid ' nitrogen, before grinding.
• a cryogenic liquid such as liquid ' nitrogen
• One particularly suitable grinding apparatus is an impact mill manufactured by Vortec Products, Long Beach, California. The Vortec impact mill allows for simultaneous introduction of both the encysted Haematococcus cells and liquid nitrogen so that the cells are cooled below the desired temperature during the grinding procedure. After grinding, the liquid nitrogen sublimates, leaving a dry final product.
• the pigment compositions of the present invention will include a variety of pigments derived from the Haematococcus cells.
• the pigments include astaxanthin esters, alpha-carotene, beta-carotene, lutein, violoxanthin, neoxanthin, chlorophyl a, chlorophyl b, and free astaxanthin, as well as trace amounts of lutein epoxide, zeaxanthin, antheraxanthin, echinenone, canthaxanthin, and various keto-carotenoids.
• Astaxanthin esters are the primary pigment in Haematococcus pluvialis cysts, typically ranging from 60% to 80% by weight of the total pigment content.
• the astaxanthin content of the pigment composition will typically be at least about 0.5% by weight based on the total product weight, usually being in the range from about 1% to 2% of the total product weight.
• the pigment compositions of the present invention will usually be " combined in a feed composition formulated for the animal to be fed.
• Such formulations typically include grains, such as wheat, alfalfa, soybean, and rice flours; fish meals; shrimp meals; as well as vitamin and oil supplements.
• grains such as wheat, alfalfa, soybean, and rice flours; fish meals; shrimp meals; as well as vitamin and oil supplements.
• a wide variety of formulations are reported in both the patent and scientific literature.
• the pigmentation compositions of the present invention may be added to such conventional feed compositions, typically at a concentration in the range from about 10 to 200 ppm, more usually in the range from about 25 to 100 ppm. Such formulations-may then be fed to the animals by conventional techniques.
• Aquatic animals which may benefit from receiving feed compositions supplemented with the pigment composition of the present invention include fish (pisces) , such as salmon, trout, and pigmented carp; crustaceans, such as shrimp, prawns, lobster and crab.
• Other animals having a desirable yellow or orange pigmentation, such as chickens may also benefit from the feed compositions of the present invention.
• the comminuted Haematococcus can also serve as a source extracted and purified carotenoids, particularly astaxanthin, which may find use in food supplements, colorants, and the like.
• Carotenoids may be extracted from the comminuted Haematococcus by conventional extraction techniques using suitable organic solvents, including oils; aromatics, e.g., benzene; halogenated hydrocarbons, e.g., methylene chloride; alkanes, e.g., hexane, and the like.
• the comminution process of the present invention is . critical in obtaining improved yields of carotenoids from Haematococcus .
• edible oils such as vegetable oils
• the resulting proeduct may be used directly as a feed supplement with minimal or no further processing.
• the comminuted cells are mixed with the solvent, and the resulting liquid phase containing the total lipid fraction (including the carotenoids) separated by filtration.
• the extracted carotenoids may also be further purified by conventional techniques, such as adsorption, chromatography, solvent-solvent extraction, crystallization, and the like.
• desired purity will be at least about 50% by weight, more usually be at least about 75% by weight, and frequently being at least about 90% by weight, and above.
• Bacteria-free, unialgal stock cultures are maintained in medium with the following composition:
• the medium is made up in deionized water and adjusted to pH 7.3. If solid medium is desired, 1.5% agar is added before autoclaving.
• the starting inoculum cultures are green, vegatative, swimming cells grown axenically in successively larger flasks until approximately the 2 liter stage. As inoculum is transferred to 10 and 200 liter clear plastic vessels for larger scale growth, the sodium acetate is no longer added to the medium and 101 mg/liter sodium nitrate is substituted for the urea. Beyond this growth stage, all of the cultures are autotrophic. Up to approximately the 200 liter stage, cultures are maintained in controlled indoor conditions. Sixteen hours of light are provided
• inoculum cultures are grown as swimming cells in outdoor ponds.
• the outdoor medium is made with irrigation water disinfected by filtration, chlorination, ozonation, or ultraviolet light. To this water are added 1.0 M ammonium bicarbonate, 0.4 mM potassium phosphate dibasic, 0.02 mM ferric chloride. 0.01 mM EDTA, and 0.025 mM magnesium sulfate.
• the outdoor culture containments are low-walled and have a center divider. They are lined with white plastic. A paddlewheel at one end produces slow circular mixing of the culture.
• the harvest method used takes advantage of the fact that mature red cysts have a density significantly greater than water. With the paddlewheel turned off, the cysts settle to the bottom of the pond within approximately one hour. The medium on top is then pumped off, resulting in an initial reduction in volume of about 80%. The cysts are further concentrated before drying. Centrifugation is a convenient means of accomplishing this. Since the cysts are quite dense and resistant to mechanical damage, many types of continuous flow centrifuges will work well. The resulting algal paste is next usually heated to approximately 70*C to kill the Haematococcus as well as any contaminating cells. The paste is next taken to near dryness (10% water or less) before grinding.
• the impact mill is set to operate at maximum speed of 20,000 rpm. Flow rates can be as high as 1.5 kg per minute. Dry ground product emerges from the mill. Due to clumping of the cells, a second pass of grinding with the same conditions is run to assure that virtually all of the cells are broken. The fine powder emerging from this second pass is final product. Fish Feed Formulation The red powder was assayed for xanthophyll content by extraction of a sample with organic solvents and spectrophoto etric reading. Total xanthophyll levels between 1.0% and 2.5% are normal.
• the red powder may be combined with an edible powder, such as o wheat flour, in order to provide a product having a consistent xanthophyll content, e.g., 1.0%.
• an edible powder such as o wheat flour
• Xanthophyll levels of between 20 and 50 ppm have been used in blending fish feed for feeding trials.
• Successful natural orange-pink coloration of Coho J salmon, sea bream and koi carp has been achieved in feeding trials.
• the pigmentation has been as good or better than that obtained by the addition of synthetic astaxanthin or synthetic canthaxanthin.

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• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
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• Engineering & Computer Science (AREA)
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• Chemical Kinetics & Catalysis (AREA)
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• Bioinformatics & Cheminformatics (AREA)
• General Engineering & Computer Science (AREA)
• General Health & Medical Sciences (AREA)
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• Fodder In General (AREA)
• Feed For Specific Animals (AREA)
• Medicines Containing Material From Animals Or Micro-Organisms (AREA)
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• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Orthopedics, Nursing, And Contraception (AREA)
• Coloring Foods And Improving Nutritive Qualities (AREA)
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• Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

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• 1988
  • 1988-02-08 US US07/153,308 patent/US4871551A/en not_active Expired - Lifetime
• 1989
  • 1989-01-20 AU AU31823/89A patent/AU605082B2/en not_active Ceased
  • 1989-01-20 WO PCT/US1989/000220 patent/WO1989006910A1/en active IP Right Grant
  • 1989-01-20 DE DE8989902666T patent/DE68905717D1/de not_active Expired - Lifetime
  • 1989-01-20 AT AT89902666T patent/ATE87428T1/de not_active IP Right Cessation
  • 1989-01-20 EP EP89902666A patent/EP0356499B1/en not_active Expired - Lifetime
  • 1989-01-20 JP JP1502478A patent/JPH02503632A/ja active Pending
  • 1989-01-25 IS IS3431A patent/IS1457B6/is unknown
  • 1989-01-26 NZ NZ227744A patent/NZ227744A/en unknown
  • 1989-02-01 CA CA000589743A patent/CA1333345C/en not_active Expired - Fee Related
  • 1989-02-06 GR GR890100069A patent/GR1000274B/el unknown
  • 1989-02-07 IE IE38989A patent/IE61202B1/en not_active IP Right Cessation
  • 1989-02-07 ES ES8900426A patent/ES2012633A6/es not_active Expired - Lifetime
  • 1989-02-08 PT PT89656A patent/PT89656B/pt active IP Right Grant
  • 1989-10-03 NO NO893927A patent/NO174274C/no unknown
  • 1989-10-05 FI FI894718A patent/FI894718A0/fi not_active IP Right Cessation
  • 1989-10-06 DK DK493989A patent/DK493989A/da not_active Application Discontinuation

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